It has previously been established that alternative, indirect pathways of formation of aminoacyl-tRNAs exist in some organisms for the amino acids glutamine, asparagine and selenocysteine. The goal of this proposal is to investigate the means by which the archaea (archaebacteria), one of the three lines of descent, generate cysteinyl- tRNA given the inability to detect cysteinyl-tRNA synthetase in archaeal genome sequences. Evidence for direct and indirect pathways will be examined by assaying cysteinyl-tRNA formation in cell extracts of the methanogenic archaea. The enzyme(s) involved in the activity will then be purified and the respective gene(s) cloned using N-terminal protein sequences. An overexpression system in E. coli will then be used to produce sufficient material for biophysical and biochemical characterizations. In particular, the RNA:protein interactions required for translational fidelity will be investigated using both site-directed mutagenesis and X-ray crystallography of the tRNA and the identified enzyme(s). The results should provide insight into the evolution of the genetic code and into the limits on the flexibility of the translational apparatus in all three domains of life.